Our genetic, molecular and biochemical analyses of ?-globin gene regulation have yielded novel and surprising results, including that the locus control region (LCR) is not required to initiate or propagate the "open" chromatin structure of the locus or confer stage-specific expression of the ?-like globin genes. In its native location, the primary role of the LCR is to enhance the transition from basal to activated transcription. We now propose to gain further insights into the molecular basis for initiating and propagating the chromatin and transcription states of the ?-globin locus during erythropoiesis. Our Specific Aims include: 1. Define epigenetic features including histone modifications, sequence co-localization, DNA methylation and candidate trans-acting factors involved in the initiation and propagation of active and silent states of the ?-globin locus. We will test the hypothesis that the ?-globin "domain" is larger and more dynamic than formerly appreciated, and that specific sequences, factors and epigenetic modifications are involved in the initiation and propagation of active and silent states of the locus. Initial studies, in collaboration with Dr. X-D Fu (UCSD) who developed the highly sensitive DNA Selection and Ligation (DSL) array-based method, will focus on a 1mb region of human chromosome 11 (h11) centered on the LCR, using a currently available array. We will design and build a similar array for analysis of the mouse locus. 2. Determine the role of cis- acting regulatory elements, transcription and cellular background on domains of histone modifications, factor binding and sequence co-localization in the ?-globin locus. We will use targeted mutation analyses in ES cell derived mice and our h11 transfer system to investigate several hypotheses regarding the regulation of p-globin gene expression. The effects of mutations on the epigenetic states of the locus, including factor binding, histone modifications and sequence interactions, will be determined by DSL analyses in stage-specific erythroid cells from mutant mice and in cell lines containing WT and modified h11s passed through different cellular backgrounds. Correlation of these results with activation state defined by primary transcript FISH, HS formation and the degree and extent of generalized DNase sensitivity will address several models of p-globin gene regulation. 3. Identify novel genes involved in erythroid maturation and p-globin gene expression. We will perform unbiased, genome-wide siRNA screens in the well- characterized G1E-ER cells and in ES derived erythroid progenitor (ES-EP) lines to identify novel factors involved in regulating p-globin gene expression and erythroid maturation. The screen makes use of a lentiviral siRNA library comprised of a redundant set of siRNAs for each known mouse gene. We propose several strategies for the validation, identification and prioritization of candidate siRNAs, as well as for the biochemical and functional analysis of genes identified.